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Investigations of Membrane Polypeptides by Solid-state NMR Spectroscopy: Structure, Dynamics, Aggregation and Topology of Supramolecular Complexes. Burkhard Bechinger Université Louis Pasteur, CNRS - UMR Chimie-physique moléculaire et spectroscopie Strasbourg, France. Solution NMR - PowerPoint PPT Presentation

Transcript of Burkhard Bechinger Universit© Louis Pasteur, CNRS - UMR

  • Investigations of Membrane Polypeptides by Solid-state NMR Spectroscopy: Structure, Dynamics, Aggregation and Topology of Supramolecular Complexes

    Burkhard Bechinger

    Universit Louis Pasteur,

    CNRS - UMR

    Chimie-physique molculaire et spectroscopie

    Strasbourg, France

  • NMR to study membrane proteins

    Solution NMR

    Requires fast and isotropic motional averaging

    < 120 kDa (TROSY)

    Solid-state NMR

    frozen, dry

    liquid crystalline membranes

    no physial size limitation

    Structure, orientation

    and dynamics

    solution and solids

  • NMR to study membrane proteins

    Solution NMR

    Requires fast and isotropic motional averaging

    < 120 kDa (TROSY)

    Extended liquid crystalline bilayers are too big

    Solid-state NMR

    frozen, dry

    liquid crystalline membranes

    no physical size limitation

    Structure, orientation

    and dynamics

    solution and solids

  • Solid-state NMR provides
    information on

    chemical environmentdistancesdihedral anglesorientations in space

    Structure, dynamics and topology

    solids

    2.unknown5.unknown9.unknown
  • Oriented membranes

    Bo

    7.unknown8.unknown
  • Chemical synthesis of peptides allows labelling at single sites

    5815.unknown
  • Oriented Samples:
    Structure and Topology

  • 15N chemical shift
    alignment of the peptide bond

    21.unknown28.unknown29.unknown30.unknown34.unknown
  • Solution and solid-state NMR on the same scale

    2723.unknown
  • The 2H quadrupolar splitting
    2H3-alanine

    Similar principles apply for many NMR interactions

    Ca

    Cb

    2H

    2H

    2H

    Bo

    Q

    DnQ ~ 3 cos2Q-1

    3679.unknown3680.unknown
  • Detailed helix alignment from
    combined 15N and 2H measurements

    2 angles

    2 measurables

    5814.unknown
  • Unique solution from Energy Minimization

    +

    hydrophobic

    hydrophilic

    +

    +

    +

    +

    +

    Tilt 95o, pitch 173o

  • KL14 Model Peptide in Oriented Phosphatidylcholine Bilayers

    Lipid2H (kHz)15N (ppm)

    POPC 6.074

    DMPC 7.673

    PC20:1 8.373

    DOPC 10.874

    Difference 2o

  • Dynamics:
    Rotational Diffusion and Aggregation

  • Liquid crystalline membranes

    Motion around the membrane normal

    5816.unknown
  • Rotational averaging:
    Effect on 15N powder pattern line shape

    Static

    Rotation around

    s33 (helix long axis)

    Rotation around

    s22

    Powder pattern provide orientational information

    5817.unknown5818.unknown5819.unknown
  • 2H solid-state NMR
    2H3-alanyl

    6322.unknown6323.unknown6324.unknown
  • Freezing Rotational Diffusion

    TM helix

    IP helix

    Loss of intensity

    during transition

    6329.unknown
  • Equilibrium: Mono- / oligomer

    2H-NMR

    6325.unknown
  • 2H NMR of real samples
    e.g. viral channel peptides

    Influenza M2 Vpu

    3686.unknown
  • 2H spectral line shape and mosaic spread

    Tilt angle:

    10o 40o 50o 700

    Mosaic

    Spread

    0.5

    1

    3

    5

    10

    15

  • Model amphipathic helix

    Q = 45.3o or 65.5o

    Mosaic spread = 1o

    3688.unknown
  • Example:
    Controlling Topology

  • Oriented 15N solid-state NMR:
    LAH4 pH-dependent molecular switch

    6516.unknown6518.unknown
  • Example: Domain of ICP47

    Herpes simplex virus87 residues early gene product
    (domain 2-34 active)Inhibits transport by TAP of antigenic peptides to surface and thus presentation by MHC I

    lack of immunogenic response

    Solution NMR:

    Helix (5-14)-loop-helix (22-31) in SDS micelles


    c/o Robert Tamp - Frankfurt

  • 15N solid-state NMR of ICP47(2-34)
    in oriented POPC

    Helix1

    Loop

    Helix2

    Modelling

    tilt 84o

    tilt 75o

  • 2H solid-state NMR of ICP47(2-34)
    in oriented POPC

    Mosaic spread 10-15o

    5952.unknown
  • Model for membrane-bound ICP47

  • Acknowledgements

    Christopher AisenbreyChristina SizunBas VogtJesus Raya


    Grard Nullans, ULP-INSERM NeurochimieRobert Tamp, Universitt Frankfurt

    ARC, ANRS, Vaincre la Mucoviscidose

    Region Alsace

    CNRS, Ministre, ACI Jeune Equipe

  • Methods to orient lipid bilayers

    Combine MAS and

    oriented samples

    5336.unknown5337.unknown
  • MAOSS at 10 kHz

    31P NMR of oriented bilayers

    10 kHz

    565 Hz

    simulated

    5339.unknown
  • MAS side band analysis provides orientational information

    5340.unknown
  • MAOSS of hydrophobic model peptide in phospholipid bilayer

    15N NMR 31P NMR

    3.7o mosaic

    20 % powder

    b=10o

    s=25o

    5341.unknown5342.unknown5343.unknown5345.unknown
  • Summary

    MAOSS with new sample set up

    low or fast spinning

    spinning side band analysis

    tilt, mosaic spread and powder pattern contributions

  • Model for membrane-bound ICP47

    6089.unknown

    33

    22

    11

    ||

    ppm50150100250200

    33

    11

    22

    ||

    22

    33

    22

    11

    33

    22

    11

    Glaubitz et al.

    250

    200

    150

    100

    50

    ppm

    Powder

    Parallel

    Perpendicular

    Experiment

    New

    B

    o

    B

    o

    b

    PD

    a

    PD

    g

    PD

    250

    200

    150

    100

    50

    ppm

    b

    PD

    a

    PD

    g

    PD

    ppm

    20

    30

    10

    0

    -20

    20 0 kHz 10 -10 -20 20 0kHz 10 -10 -20

    50 0kHz313 K303 K293 K283 K263 K

    L

    5

    E

    6

    M

    7

    A

    8

    D

    9

    T

    10

    F

    11

    L

    12

    D

    13

    N

    14

    L

    5

    E

    6

    M

    7

    A

    8

    D

    9

    T

    10

    F

    11

    L

    12

    D

    13

    N

    14

    helix 1

    Y

    22

    A

    23

    D

    24

    V

    25

    V

    25

    R

    26

    A,N

    27

    E

    28

    I

    29

    N

    30

    K

    31

    Y

    22

    A

    23

    D

    24

    R

    26

    A,N

    27

    E

    28

    I

    29

    N

    30

    K

    31

    helix 2

    1422 5

    31

    pH > 7

    pH < 5

    pH 6.1

    Deblock

    Activate aa

    Couple

    resin

    resin

    resin

    resin

    to

    reactor

    Wash

    Wash

    255075100125150175

    25

    5075

    100

    125

    150

    175

    rotational pitch angle tilt angle

    0

    30

    5390

    60

    ppm

    20 0kHz 10 -10

    20 0kHz -20 20 0kHz

    20 0 kHz 10 -10 -20